Mesoscale sequence feature modulates AID activity in antibody diversification

The adaptive immune system produces diverse antibodies against a wide range of invading pathogens. Three mechanisms contribute to antibody diversification [1]. During early B cell development, V(D)J recombination assembles immunoglobulin(Ig) genes by joining the variable (V), diversity (D) and joining (J) gene segments to form a large primary antibody repertoire. After antigenic challenge, activated B cells perform class switch recombination (CSR) to change the Ig heavy chain constant regions (IgH) to expand the scope of immune response. Concurrent with CSR, somatic hypermutation (SHM) modifies Ig heavy and light chain variable regions (IgV) to improve antigen-binding affinity and produce high-affinity antibodies [2]. Both CSR and SHM occur in activated germinal center B cells and are initiated by activation-induced cytidine deaminase (AID) [3]. The mutagenic activity of AID is mainly confined to switch (S) regions and IgV regions. SHM introduces further mutations in the IgV exons to allow affinity maturation, which is at the center of the humoral immune response to counter infections [2]. Why these mutations are largely concentrated in the non-consecutive complementarity-deter-mining regions (CDRs) of IgV remains an enigma [4]. The groundbreaking study by Wang and colleagues [5],recently published in Cell, shed some light on this

The adaptive immune system produces diverse antibodies against a wide range of invading pathogens.Three mechanisms contribute to antibody diversification [1].During early B cell development, V(D)J recombination assembles immunoglobulin (Ig) genes by joining the variable (V), diversity (D) and joining (J) gene segments to form a large primary antibody repertoire.After antigenic challenge, activated B cells perform class switch recombination (CSR) to change the Ig heavy chain constant regions (IgH) to expand the scope of immune response.Concurrent with CSR, somatic hypermutation (SHM) modifies Ig heavy and light chain variable regions (IgV) to improve antigen-binding affinity and produce highaffinity antibodies [2].
Both CSR and SHM occur in activated germinal center B cells and are initiated by activation-induced cytidine deaminase (AID) [3].The mutagenic activity of AID is mainly confined to switch (S) regions and IgV regions.SHM introduces further mutations in the IgV exons to allow affinity maturation, which is at the center of the humoral immune response to counter infections [2].Why these mutations are largely concentrated in the non-consecutive complementarity-determining regions (CDRs) of IgV remains an enigma [4].The groundbreaking study by Wang and colleagues [5], recently published in Cell, shed some light on this long-standing question that has puzzled antibody researchers for more than 40 years.It demonstrats that the flexible single-strand DNA (ssDNA) feature is the key to determine preferential hypermutability by increasing binding to the surface-charged patches of AID and facilitating deaminase activity.

CDR Hypermutation Is an Evolutionary-conserved Feature Influenced by AID Activity and DNA Sequence per se
The CDRs are three short spaced hypervariable intervals located in the IgV exons, which are flanked by four relatively stable regions, termed as framework regions (FRs).The CDRs undergo a higher frequency of mutations than the FRs (Figure 1A).This CDRpreferential hypermutation feature mainly relies on the predisposition of mutations, as evidenced by similar patterns observed in the intrinsic hypermutation profiles from non-productive V exons [4].While the analysis of SHM hotspots has demonstrated the preferential targeting of cytidines (Cs) by AID in the context of WRCY or the complementary RGYW motif (W=A/T, R=A/G, Y=C/T), it is important to note that only a limited number of IgV sequences have been examined for their intrinsic SHM profiles [6,7].In a recent paper published in Cell, by utilizing a vast database of SHM profiles of the non-functional Ig heavy chain variable region (IgV H ) sequences, Wang et al. [5] demonstrated that the CDRs exhibit a great abundance of WRC motifs than the FRs.Crucially, they also established that cytidines in WRC motifs undergo mutations at a higher frequency in CDRs than in FRs.
To explore the molecular determinants behind CDR preference, Wang et al. [5] constructed an in vitro AID deamination assay containing purified AID and ssDNA substrates that performs mimicable SHM profiles comparing to in vivo hypermutation patterns.They found that the in vivo CDR-preferential hypermutation profiles could be recapitulated in an in vitro AID deamination assay on ssDNA.This remarkable discovery demonstrated that both AID and ssDNA sequence per se possess the ability to determine preferential hypermutability of WRC in CDR-vs.-FR.Furthermore, Wang et al. [5] extended the biochemical assay to analyze in vitro AID deamination profiles from 27 species, and they successfully established that the CDR-preferential hypermutability is highly evolutionary-conserved in tetrapod species.However, intriguingly, this general trend does not hold true for horses and GALT (gut-associated lymphoid tissue) species.In addition, Wang et al. [5] further demonstrated that the mutation frequencies of WRC in CDR3s are higher than that in the CDR1/2s, although the sequence features of CDR3 are either inherited from D/J segments or acquired through V(D)J recombination.

DNA Sequence Affects SHM through Electrostatic Interactions between Mesoscale Adjacent Sequence of WRC Motifs and AID
An intricate network is applied to precisely regulate the efficacy of AID due to its DNA-damaging potential.While regulatory mechan-isms are manipulated across multiple scales, ranging from nuclear large-scale to WRC motif microscale, the mesoscale level (5-50 bp) has not received adequate attention [8].Using the powerful passenger-Ig mouse model system [4], Wang et al. [5] demonstrated that WRC surrounding sequence contexts contribute to the WRC mutability in vivo, yet the WRC position per se does not determine mutability.They extended the passenger-Ig mouse model system by integrating CRISPR/Cas9-mediated CDR3 editing.Through the analysis of the SHM profiles in these models, they successfully established that the mesoscale sequence surrounding the WRC motifs contributes to the adjacent WRC mutability in vivo and plays a direct role in regulating AID deaminase activity at the ssDNA level.
AID interacts with ssDNA via a bifurcated substrate-binding surface, capturing two structured adjacent ssDNAs, one of which is identified as a substrate channel and the other as a assistant patch [9].Wang et al. [5] hypothesized that AID could "sense" the mesoscale sequence feature adjacent WRC motifs via these surface patches.Using a combination of in vitro AID deamination assay, molecular dynamics simulations and single-molecule biochemistry, they demonstrated that AID surface patch-mediated interaction may drive the mesoscale preference.This interaction could potentially play a role in determining the preferential deamination based on electrostatic interactions between AID and ssDNA backbone.Moreover, the ssDNA base sequence might indirectly impact the binding process.

DNA Strand Flexibility Contributes AID Deamination Preference in a Non-coding Way
DNA flexibility is a sequence-dependent conformational property.Previous reports showed that poly(dA) exhibits high rigidity, while poly(dT) displays remarkable flexibility [10].The use of homopolymer-context substrates allows for the examination of how mesoscale DNA flexibility influences AID activity.In their study, Wang et al. [5] conducted experiments using a panel of substrates with increasing rigidity on either side of the AGCT motif, and demonstrated that the DNA region immediately 5′ to the AID target directly binds to AID via the charged patch.They also established that AID-favored substrates, which are enriched in pyrimidinepyrimidine (Py-Py) motifs and characterized by weak stacking strength and high flexibility, demonstrate high mutability; whereas AID-disfavored substrates, which are enriched in purine-purine (Pu-Pu) motifs and have strong stacking strength and low flexibility, exhibit low mutability (Figure 1B).During the evolution of antibody genes, the DNA sequence encoding the CDRs has acquired the feature of high flexibility, and insertion of a synthetic flexible motif adjacent to a cold-WRC motif renders it highly mutable [5].Collectively, the study revealed that the AID sequence preference is modulated by the ssDNA flexibility, uncovering a non-coding role of the mesoscale DNA sequences in the CDRs in promoting SHM (Figure 1B) and offering insights into the underlying mechanisms behind hypermutation patterns observed in B cell lymphoma.
In summary, Meng and colleagues [5] have successfully solved a long-standing question in the field of SHM.Their findings have profound implications for our understanding of the intricate mechanisms underlying IgV sequence evolution and AID non-Ig off-targeting preference.These discoveries provide valuable new insights into the future development of advanced humanized antibody animal models.Nevertheless, several intriguing questions still warrant further consideration.These include: 1) understanding the acquisition process of the mesoscale feature in CDR of IgV during evolution; 2) unraveling the unresolved mechanisms behind the acquisition of these features in somatic-assembled CDR3; 3) elucidating the influence of DNA secondary structure on flexibility in vivo; and 4) obtaining additional genetic evidence to comprehend the impact of the mesoscale feature on AID-initiated genome instability in cancer.

Figure 1 .
Figure 1.Mesoscale DNA feature facilitates somatic hypermutation (A) An illustrative artificial dataset shows the higher mutation frequency in WRC motifs in CDR-vs.-FR.(B) Model for flexible ssDNA feature in facilitating AID activity.Flexible pyrimidine-pyrimidine (Py-Py) bases frequently flank WRC motifs in CDR.Wang et al. [5] found that mesoscale DNA sequences exhibit strong binding affinity to the positively charged surface patches of AID, resulting in a preferential enhancement of deamination activity.